The functionality of natural killer (NK) cells is tuned during education and is associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation in killer cell immunoglobulin-like receptor (KIR) and HLA, which is known to influence the functional strength of NK cells, fine-tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but were stable over time in each individual and genetically determined by allelic variation in HLA class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of major histocompatibility complex-deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells, resulting in predictable hierarchies in global NK cell function.

Miniaturization of cell culture substrates enables controlled analysis of living cells in confined micro-scale environments. This is particularly suitable for imaging individual cells over time, as they can be monitored without escaping the imaging field-of-view (FoV). Glass materials are ideal for most microscopy applications. However, with current methods used in life sciences, glass microfabrication is limited in terms of either freedom of design, quality, or throughput. In this work, we introduce laser-induced deep etching (LIDE) as a method for producing glass microwell arrays for live single cell imaging assays. We demonstrate novel microwell arrays with deep, high-aspect ratio wells that have rounded, dimpled or flat bottom profiles in either single-layer or double-layer glass chips. The microwells are evaluated for microscopy-based analysis of long-term cell culture, clonal expansion, laterally organized cell seeding, subcellular mechanics during migration and immune cell cytotoxicity assays of both adherent and suspension cells. It is shown that all types of microwells can support viable cell cultures and imaging with single cell resolution, and we highlight specific benefits of each microwell design for different applications. We believe that high-quality glass microwell arrays enabled by LIDE provide a great option for high-content and high-resolution imaging-based live cell assays with a broad range of potential applications within life sciences. 

Glass materials have excellent optical and chemical properties for microscopy-based live cell assays but state-of-the-art methods for microfabrication of Lab-on-Chip (LoC) devices are often limited by either complex manufacturing and/or low quality results. In this work, we have evaluated glass microwell array chips produced using a recently introduced laser-based microfabrication method. Three different types of microwell designs have been tested for imaging and screening of on-chip cell cultures and live cell assays.

Immune synapses are large-scale, transient molecular assemblies that serve as platforms for antigen presentation to B and T cells and for target recognition by cytotoxic T cells and natural killer (NK) cells. The formation of an immune synapse is a tightly regulated, stepwise process in which the cytoskeleton, cell surface receptors, and intracellular signaling proteins rearrange into supramolecular activation clusters (SMACs). We generated artificial immune synapses (AIS) consisting of synthetic and natural ligands for the NK cell-activating receptors LFA-1 and CD16 by microcontact printing the ligands into circular-shaped SMAC structures. Live-cell imaging and analysis of fixed human NK cells in this reductionist system showed that the spatial distribution of activating ligands influenced the formation, stability, and outcome of NK cell synapses. Whereas engagement of LFA-1 alone promoted synapse initiation, combined engagement of LFA-1 and CD16 was required for the formation of mature synapses and degranulation. Organizing LFA-1 and CD16 ligands into donut-shaped AIS resulted in fewer long-lasting, symmetrical synapses compared to dot-shaped AIS. NK cells spreading evenly over either AIS shape exhibited similar arrangements of the lytic machinery. However, degranulation only occurred in regions containing ligands that therefore induced local signaling, suggesting the existence of a late checkpoint for degranulation. Our results demonstrate that the spatial organization of ligands in the synapse can affect its outcome, which could be exploited by target cells as an escape mechanism.

Natural killer (NK) cells are innate immune cells with the ability to recognize and eliminate virally infected cells and cancer cells without prior sensitization. There is a functional heterogeneity between individual NK cells, where some NK cells are more efficient at killing cancer cells than others. Methods that allow studies of single NK cells are required to understand the functional differences and how they correlate with the activation and development status of the NK cell.

This thesis focuses on the development and implementation of microchip- based imaging of NK cells, which is covered in five papers. Paper I presents a microchip screening platform for assessment of the cytotoxic potential of individual NK cells, by confining single NK cells together with target cells in microwells, followed by microscopy screening over extended time periods and automated image analysis. In paper II, the microchip platform was applied to test the ability of a novel trispecific killer engager (TriKE) to mediate an NK cell-dependent immune response. The process of NK cell education was studied in paper III and for that the image analysis methods for the microchip platform was further developed, in order to reveal new insight into how the education process affects the cytotoxic function of single NK cells. In paper IV, a previously developed microchip assay was extended to study NK cell migration and cytotoxicity in a more in vivo-like 3D collagen matrix. Paper V shows how NK cells can eliminate platelets in the presence of anti-platelet antibodies.

In summary, this thesis covers the development and applications of time- lapse imaging using microwells for studying important NK cell functions in different settings. Understanding NK cell heterogeneity has the potential for improving e.g. cancer cell therapies.

NK-celler tillhör det ospecifika immunförsvaret och har som uppgift att hitta och eliminera virusinfekterade celler och tumörceller. Det har visat sig att NK-celler är funktionellt heterogena, vilket leder till att vissa NK- celler dödar tumörceller mer effektivt än andra. För att förstå dessa funktionella skillnader och hur de är kopplade till t.ex. cellernas mognad och aktivering, krävs metoder som gör det möjligt att studera NK-celler på encellsnivå.

Denna avhandling fokuserar på utveckling och implementering av mikrochipbaserad avbildning av NK-celler, vilket behandlas i fem artiklar. I artikel I presenteras en mikrochipplattform som används för att studera den cytotoxiska potentialen hos enskilda NK-celler. Detta genom att fånga upp enstaka NK-celler tillsammans med tumörceller i tiotusentals mikrobrunnar och därefter avbilda dem i mikroskop under längre tider med efterföljande automatiserad bildanalys. I artikel II används mikrochipplattformen för att studera funktionen hos en nyutvecklad molekyl (TriKE), som både binder till tumörcellen samtidigt som den aktiverar NK-cellen. I artikel III används mikrochipplattformen för att studera en viktig process där NK celler ”utbildas” för att lära sig skilja mellan tumörceller och normala celler. I artikel IV används en större variant av mikrobrunnarna för att kunna återskapa en in vivo-liknande miljö för NK-cellerna genom att bädda in dem i en tredimensionell kollagenmatris. Artikel V visar hur NK-celler kan eliminera blodplättar i närvaro av antikroppar.

Sammanfattningsvis handlar denna avhandling om utveckling och tillämpningar av mikroskopisk avbildning av levande NK-celler med hjälp av mikrobrunnar i syfte att studera dess viktiga funktioner under olika förhållanden. Ökad kunskap om NK-cellernas heterogenitet har potentialen att förbättra effektiviteten hos t.ex. cellterapier.

Natural killer (NK) cell cytotoxicity in tissue is dependent on the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment. Traditional imaging studies of NK cell migration and cytotoxicity have utilized 2D surfaces, which do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. Here, we have combined a microwell assay that allows long-term imaging and tracking of small, well-defined populations of NK cells with an interstitial ECM-like matrix. The assay allows for long-term imaging of NK-target cell interactions within a confined 3D volume. We found marked differences in motility between individual cells with a small fraction of the cells moving slowly and being confined to a small volume within the matrix, while other cells moved more freely. A majority of NK cells also exhibited transient variation in their motility, alternating between periods of migration arrest and movement. The assay could be used as a complement to in vivo imaging to study human NK cell heterogeneity in migration and cytotoxicity.

Myelodysplastic syndrome (MDS) is a clonal heterogeneous stem cell disorder driven by multiple genetic and epigenetic alterations resulting in ineffective hematopoiesis. MDS has a high frequency of immune suppressors, including myeloid-derived suppressor cells (MDSCs), that collectively result in a poor immune response. MDSCs in MDS patients express CD155 that ligates the T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) and delivers an inhibitory signal to natural killer (NK) cells. To mediate a productive immune response against MDS, negative regulatory checkpoints, like TIGIT, expressed on MDS NK cells must be overcome. NK cells can be directed to lyse MDS cells by bispecific killer engagers (BiKEs) that ligate CD16 on NK cells and CD33 on MDS cells. However, such CD16 x CD33 (1633) BiKEs do not induce the proliferative response in MDS NK cells needed to sustain their function. Here, we show that the addition of an NK stimulatory cytokine, interleukin-15 (IL-15), into the BiKE platform leads to productive IL-15 signaling without TIGIT upregulation on NK cells from MDS patients. Lower TIGIT expression allowed NK cells to resist MDSC inhibition. When compared with 1633 BiKE, 161533 trispecific killer engager (TriKE)-treated NK cells demonstrated superior killing kinetics associated with increased STAT5 phosphorylation. Furthermore, 161533 TriKE-treated MDS NK cells had higher proliferation and enhanced NK-cell function than 1633 BiKE-treated cells without the IL-15 linker. Collectively, our data demonstrate novel characteristics of the 161533 TriKE that support its application as an immunotherapeutic agent for MDS patients.

Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19(+) targets, and maintained their intrinsic degranulation response toward CD19(-) K562 cells. The response of redirected NK-cell subsets against CD19(+) targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA-specific KIR(s), displayed superior ability to kill CD19(+), HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells.